Cutting Edge: Aire Is a Coactivator of the Vitamin D Receptor.

Vitamin D deficiency is associated with the development of autoimmunity, which arises from defects in T cell tolerance to self-antigens. Interactions of developing T cells with medullary thymic epithelial cells, which express tissue-restricted Ags, are essential for the establishment of central tolerance. However, vitamin D signaling in the thymus is poorly characterized. We find that stromal and hematopoietic cells in the mouse thymus express the vitamin D receptor (Vdr) and Cyp27b1, the enzyme that produces hormonal 1,25-dihydroxyvitamin D (1,25D). Treatment of cultured thymic slices with 1,25D enhances expression of the critical medullary thymic epithelial cell transcription factor autoimmune regulator (Aire), its colocalization with the Vdr, and enhances tissue-restricted Ag gene expression. Moreover, the Vdr interacts with Aire in a 1,25D-dependent manner and recruits Aire to DNA at vitamin D response elements, where it acts as a Vdr coactivator. These data link vitamin D signaling directly to critical transcriptional events necessary for central tolerance.

Type I interferon induced during chronic viral infection favors B-cell development in the thymus.

Chronic viral infections cause thymic involution yet the potential for broader, longer-term impact on thymic composition remains unexplored. Here we show that chronic, but not acute, lymphocytic choriomeningitis virus infection promotes a unique population of immature B cells in the thymus. We show that chronic viral infection promotes signals within the thymus, including the expression of B-cell activating factor (BAFF), that favor the maturation of this population as these cells acquire expression of CD19 and immunoglobulin M. Mechanistically, type I interferon (IFN-I), predominantly IFNß, signals to thymic hematopoietic cells, strongly delaying T-cell development at the earliest precursor stage. Furthermore, IFN-I signaling to the nonhematopoietic compartment provides a second signal essential to favor B-cell differentiation and maturation within the thymus. Importantly, chronic infection yields changes in the B-cell population for at least 50 days following infection, long after thymic atrophy has subsided. Thus, the inflammatory milieu induced by chronic viral infection has a profound, and long-lasting, effect on thymic composition leading to the generation of a novel population of thymic B cells.

NR4A3 Mediates Thymic Negative Selection.

Central tolerance aims to limit the production of T lymphocytes bearing TCR with high affinity for self-peptide presented by MHC molecules. The accumulation of thymocytes with such receptors is limited by negative selection or by diversion into alternative differentiation, including T regulatory cell commitment. A role for the orphan nuclear receptor NR4A3 in negative selection has been suggested, but its function in this process has never been investigated. We find that Nr4a3 transcription is upregulated in postselection double-positive thymocytes, particularly those that have received a strong selecting signal and are destined for negative selection. Indeed, we found an accumulation of cells bearing a negative selection phenotype in NR4A3-deficient mice as compared with wild-type controls, suggesting that Nr4a3 transcriptional induction is necessary to limit accumulation of self-reactive thymocytes. This is consistent with a decrease of cleaved caspase-3+-signaled thymocytes and more T regulatory and CD4+Foxp3-HELIOS+ cells in the NR4A3-deficient thymus. We further tested the role for NR4A3 in negative selection by reconstituting transgenic mice expressing the OVA Ag under the control of the insulin promoter with bone marrow cells from OT-I Nr4a3 +/+ or OT-I Nr4a3 -/- mice. Accumulation of autoreactive CD8 thymocytes and autoimmune diabetes developed only in the absence of NR4A3. Overall, our results demonstrate an important role for NR4A3 in T cell development.

Low rates of serious complications after open Latarjet procedure at short-term follow-up.

PURPOSE: To report on intraoperative and short-term postoperative adverse events after open Latarjet procedure in patients with recurrent anterior shoulder instability. These complications were classified into different grades of severity based on the treatment required and the learning curve of the procedure. METHODS: Ninety-six patients (102 shoulders) underwent open Latarjet procedure for recurrent post-traumatic anterior glenohumeral instability between 2012 and 2020. The minimum duration of patients' follow-up was 6 months. Adverse events were classified into 3 classes based on the severity and subsequent treatment. The complications in the first 50% of all cases were compared with the latter 50% to evaluate the role of learning curve on the complication rates. RESULTS: The mean follow-up was 7.2 ± 2.8 months. The patients' mean age was 26.7 ± 8.9 years and consisted of 83 (86.4%) male and 13 (13.6%) female patients. The total adverse events rate was 18.6%. Adverse events requiring no additional treatment (class 1) occurred in 6 cases (5.8%) including fibrous union (3.9%) and asymptomatic resorption of the graft (1.9%). Adverse events requiring additional or extended nonoperative management (class 2) occurred in 8 cases (7.8%), including coracoid fracture (2.9%), musculocutaneous nerve palsy (1.9%), axillary nerve palsy (0.9%), suprascapular nerve palsy (0.9%), and stiffness (0.9%). All the nerve palsies recovered without long-term sequelae. Adverse events requiring secondary operative procedures (class 3) occurred in 5 cases (4.9%), including symptomatic hardware (1.9%), medial healing of the graft (0.9%), screw loosening (0.9%), and deep infection (0.9%). The rate of adverse events in revision cases was higher than primary cases in 11.7% and 6.8%, respectively (P = .119). The complication rate was significantly higher in the first half of the surgeons' practice (14.7%) than in the second half (3.9%) (P ≤ .05). CONCLUSIONS: The overall complication rate reported in this open Latarjet series is 18.6%; however, the rate of class 3 adverse events that required additional surgery or long-term medical treatment was only 4.9%. Revision cases had a higher rate of complications than primary cases, and the learning curve has had a significant impact on the rate of adverse events.

The Effect of Staged Versus Usual Care Physiotherapy on Knee Function Following Anterior Cruciate Ligament Reconstruction.

CONTEXT: The long duration and high cost of anterior cruciate ligament reconstruction (ACLR) rehabilitation can pose barriers to completing rehabilitation, the latter stages of which progress to demanding sport-specific exercises critical for a safe return to sport. A staged approach shifting in-person physiotherapy sessions to later months of recovery may ensure patients undergo the sport-specific portion of ACLR rehabilitation. Design/Objective: To compare postoperative outcomes of knee function in patients participating in a staged ACLR physiotherapy program to patients participating in usual care physiotherapy through a randomized controlled trial. METHODS: One hundred sixty-two patients were randomized to participate in staged (n = 80) or usual care physiotherapy (n = 82) following ACLR and assessed preoperatively and postoperatively at 2 weeks, 6 weeks, 3 months, and 6 months. The staged group completed the ACLR rehabilitation protocol at home for the first 3 months, followed by usual care in-person sessions. The usual care group completed in-person sessions for their entire rehabilitation. Outcome measures included the Lower Extremity Functional Scale, International Knee Documentation Committee Questionnaire, pain, range of motion, strength, and hop testing. RESULTS: There were no statistically significant between-group differences in measures of knee function at 6 months postoperative. Patients in the usual care group reported significantly higher International Knee Documentation Committee scores at 3 months postoperative (mean difference = 5.8; 95% confidence interval,  1.3 to 10.4; P = .01). CONCLUSION: A staged approach to ACLR rehabilitation does not appear to impede knee function at 6 months postoperative but may result in worse patient reported outcomes at early follow-ups.

CD5 levels define functionally heterogeneous populations of naïve human CD4+ T cells.

Studies in murine models show that subthreshold TCR interactions with self-peptide are required for thymic development and peripheral survival of naïve T cells. Recently, differences in the strength of tonic TCR interactions with self-peptide, as read-out by cell surface levels of CD5, were associated with distinct effector potentials among sorted populations of T cells in mice. However, whether CD5 can also be used to parse functional heterogeneity among human T cells is less clear. Our study demonstrates that CD5 levels correlate with TCR signal strength in human naïve CD4+ T cells. Further, we describe a relationship between CD5 levels on naïve human CD4+ T cells and binding affinity to foreign peptide, in addition to a predominance of CD5hi T cells in the memory compartment. Differences in gene expression and biases in cytokine production potential between CD5lo and CD5hi naïve human CD4+ T cells are consistent with observations in mice. Together, these data validate the use of CD5 surface levels as a marker of heterogeneity among human naïve CD4+ T cells with important implications for the identification of functionally biased T- cell populations that can be exploited to improve the efficacy of adoptive cell therapies.

The ICOS-ICOSL pathway tunes thymic selection.

Negative selection of developing T cells plays a significant role in T-cell tolerance to self-antigen. This process relies on thymic antigen-presenting cells which express both self-antigens and cosignaling molecules. Inducible T-cell costimulator (ICOS) belongs to the CD28 family of cosignaling molecules and binds to ICOS ligand (ICOSL). The ICOS signaling pathway plays important roles in shaping the immune response to infections, but its role in central tolerance is less well understood. Here we show that ICOSL is expressed by subsets of thymic dendritic cells and medullary thymic epithelial cells as well as thymic B cells. ICOS expression is upregulated as T cells mature in the thymus and correlates with T-cell receptor signal strength during thymic selection. We also provide evidence of a role for ICOS signaling in mediating negative selection. Our findings suggest that ICOS may fine-tune T-cell receptor signals during thymic selection contributing to the generation of a tolerant T-cell population.

MHC-Independent Thymic Selection of CD4 and CD8 Coreceptor Negative αß T Cells.

It is becoming increasingly clear that unconventional T cell subsets, such as NKT, γδ T, mucosal-associated invariant T, and CD8αα T cells, each play distinct roles in the immune response. Subsets of these cell types can lack both CD4 and CD8 coreceptor expression. Beyond these known subsets, we identify CD4-CD8-TCRαß+, double-negative (DN) T cells, in mouse secondary lymphoid organs. DN T cells are a unique unconventional thymic-derived T cell subset. In contrast to CD5high DN thymocytes that preferentially yield TCRαß+ CD8αα intestinal lymphocytes, we find that mature CD5low DN thymocytes are precursors to peripheral DN T cells. Using reporter mouse strains, we show that DN T cells transit through the immature CD4+CD8+ (double-positive) thymocyte stage. Moreover, we provide evidence that DN T cells can differentiate in MHC-deficient mice. Our study demonstrates that MHC-independent thymic selection can yield DN T cells that are distinct from NKT, γδ T, mucosal-associated invariant T, and CD8αα T cells.

CD5 levels reveal distinct basal T-cell receptor signals in T cells from non-obese diabetic mice.

Type 1 diabetes in non-obese diabetic (NOD) mice occurs when autoreactive T cells eliminate insulin producing pancreatic ß cells. While extensively studied in T-cell receptor (TCR) transgenic mice, the contribution of alterations in thymic selection to the polyclonal T-cell pool in NOD mice is not yet resolved. The magnitude of signals downstream of TCR engagement with self-peptide directs the development of a functional T-cell pool, in part by ensuring tolerance to self. TCR interactions with self-peptide are also necessary for T-cell homeostasis in the peripheral lymphoid organs. To identify differences in TCR signal strength that accompany thymic selection and peripheral T-cell maintenance, we compared CD5 levels, a marker of basal TCR signal strength, on immature and mature T cells from autoimmune diabetes-prone NOD and -resistant B6 mice. The data suggest that there is no preferential selection of NOD thymocytes that perceive stronger TCR signals from self-peptide engagement. Instead, NOD mice have an MHC-dependent increase in CD4+ thymocytes and mature T cells that express lower levels of CD5. In contrast, T cell-intrinsic mechanisms lead to higher levels of CD5 on peripheral CD8+ T cells from NOD relative to B6 mice, suggesting that peripheral CD8+ T cells with higher basal TCR signals may have survival advantages in NOD mice. These differences in the T-cell pool in NOD mice may contribute to the development or progression of autoimmune diabetes.

Differential interferon-gamma production potential among naïve CD4+ T cells exists prior to antigen encounter.

Individual CD4+ T cells can become one of a number of helper (Th) lineages with distinct effector functions. However, whether biases in Th potential exist prior to antigen encounter is unknown. Studies have identified cell-intrinsic functional heterogeneity among naïve T cells that can be parsed based on the strength of T-cell receptor (TCR) interactions with self-peptide. Here, using CD5 levels as a surrogate for the strength of these basal TCR signals, we sought to identify pre-existing effector biases in the CD4+ T-cell lineage. We show that ex vivo-activated CD5lo CD4+ T cells produce greater amounts of the Th1 cytokine interferon-gamma (IFNγ) than their CD5hi counterparts. In addition, a greater percentage of CD5lo effector CD4+ T cells produce IFNγ in both polyclonal and monoclonal CD4+ T-cell populations after antigen challenge in vivo. These results suggest that differential IFNγ production potential exists among CD4+ T cells prior to activation and independent of TCR affinity for foreign antigen.

Complement Component 3 Regulates IFN-α Production by Plasmacytoid Dendritic Cells following TLR7 Activation by a Plant Virus-like Nanoparticle.

The increasing use of plant viruses for the development of new vaccines and immunotherapy approaches poses questions regarding the mechanism by which the mammalian immune system recognizes these viruses. For example, although natural Abs (NA) and complement are key components of the innate immune system involved in the opsonization, phagocytosis, and destruction of microorganisms infecting mammals, their implication in plant virus recognition and immunogenicity is not well defined. In this study, we address the involvement of NA and the complement system in the activation of innate immunity through engagement of TLR7 with papaya mosaic virus (PapMV)-like nanoparticles. We demonstrate that NA, although binding to PapMV, are not involved in its recognition by the immune system. On the other hand, C3 strongly binds to PapMV nanoparticles and its depletion significantly reduces PapMV's interaction with immune cells. Unexpectedly, however, we observed increased immune cell activation following administration of PapMV to complement-depleted mice. TLR7 activation by PapMV in the absence of C3 induced higher IFN-α production, resulting in superior immune cell activation and increased immunotherapeutic properties. In conclusion, in this study we established the involvement of the complement system in the recognition and the phagocytosis of PapMV nanoparticles and identified an unsuspected role for C3 in regulating the production of IFN-α following TLR7 activation.

A randomised trial of observational learning from 2D and 3D models in robotically assisted surgery.

BACKGROUND: Advances in 3D technology mean that both robotic surgical devices and surgical simulators can now incorporate stereoscopic viewing capabilities. While depth information may benefit robotic surgical performance, it is unclear whether 3D viewing also aids skill acquisition when learning from observing others. As observational learning plays a major role in surgical skills training, this study aimed to evaluate whether 3D viewing provides learning benefits in a robotically assisted surgical task. METHODS: 90 medical students were assigned to either (1) 2D or (2) 3D observation of a consultant surgeon performing a training task on the daVinci S robotic system, or (3) a no observation control, in a randomised parallel design. Subsequent performance and instrument movement metrics were assessed immediately following observation and at one-week retention. RESULTS: Both 2D and 3D groups outperformed no observation controls following the observation intervention (ps < 0.05), but there was no difference between 2D and 3D groups at any of the timepoints. There was also no difference in movement parameters between groups. CONCLUSIONS: While 3D viewing systems may have beneficial effects for surgical performance, these results suggest that depth information has limited utility during observational learning of surgical skills in novices. The task constraints and end goals may provide more important information for learning than the relative motion of surgical instruments in 3D space.

Energy-Based Metrics for Arthroscopic Skills Assessment.

Minimally invasive skills assessment methods are essential in developing efficient surgical simulators and implementing consistent skills evaluation. Although numerous methods have been investigated in the literature, there is still a need to further improve the accuracy of surgical skills assessment. Energy expenditure can be an indication of motor skills proficiency. The goals of this study are to develop objective metrics based on energy expenditure, normalize these metrics, and investigate classifying trainees using these metrics. To this end, different forms of energy consisting of mechanical energy and work were considered and their values were divided by the related value of an ideal performance to develop normalized metrics. These metrics were used as inputs for various machine learning algorithms including support vector machines (SVM) and neural networks (NNs) for classification. The accuracy of the combination of the normalized energy-based metrics with these classifiers was evaluated through a leave-one-subject-out cross-validation. The proposed method was validated using 26 subjects at two experience levels (novices and experts) in three arthroscopic tasks. The results showed that there are statistically significant differences between novices and experts for almost all of the normalized energy-based metrics. The accuracy of classification using SVM and NN methods was between 70% and 95% for the various tasks. The results show that the normalized energy-based metrics and their combination with SVM and NN classifiers are capable of providing accurate classification of trainees. The assessment method proposed in this study can enhance surgical training by providing appropriate feedback to trainees about their level of expertise and can be used in the evaluation of proficiency.

Potentiating Cancer Immunotherapy Using Papaya Mosaic Virus-Derived Nanoparticles.

The recent development of novel immunotherapies is revolutionizing cancer treatment. These include, for example, immune checkpoint blockade, immunomodulation, or therapeutic vaccination. Although effective on their own, combining multiple approaches will most likely be required in order to achieve the maximal therapeutic benefit. In this regard, the papaya mosaic virus nanoparticle (PapMV) has shown tremendous potential as (i) an immunostimulatory molecule, (ii) an adjuvant, and (iii) a vaccine platform through its intrinsic capacity to activate the innate immune response in an IFN-α-dependent manner. Here, we demonstrate that intratumor administration of PapMV significantly slows down melanoma progression and prolongs survival. This correlates with enhanced chemokine and pro-inflammatory-cytokine production in the tumor and increased immune-cell infiltration. Proportions of total and tumor-specific CD8(+) T cells dramatically increase following PapMV treatment whereas those of myeloid-derived suppressor cells (MDSC) concomitantly decrease. Moreover, systemic PapMV administration prevents metastatic tumor-implantation in the lungs. Importantly, PapMV also synergistically improves the therapeutic benefit of dendritic cell (DC)-based vaccination and PD-1 blockade by potentiating antitumor immune responses. This study illustrates the immunostimulatory potential of a plant virus-derived nanoparticle for cancer therapy either alone or in conjunction with other promising immunotherapies in clinical development.

Nanoparticle adjuvant sensing by TLR7 enhances CD8+ T cell-mediated protection from Listeria monocytogenes infection.

Developing new adjuvants and vaccination strategies is of paramount importance to successfully fight against many life-threatening infectious diseases and cancer. Very few adjuvants are currently authorized for human use, and these mainly stimulate a humoral response. However, specific Abs are not sufficient to confer protection against persisting infections or cancer. Therefore, development of adjuvants and immunomodulators able to enhance cell-mediated immune responses represents a major medical need. We recently showed that papaya mosaic virus nanoparticles (PapMV), self-assembled from the coat protein of a plant virus and a noncoding ssRNA molecule, are highly immunogenic in mice. PapMV can be used either as a vaccine delivery platform, through fusion of various epitopes to the coat protein or as adjuvant to enhance humoral immune responses against coadministered Ags or vaccines. However, the mechanisms that confer these immunomodulatory properties to PapMV and its ability to enhance T cell vaccines remain unknown. Using immunization studies in mice, we demonstrate in this paper that PapMV represents a novel TLR7 agonist with strong immunostimulatory properties. More importantly, pretreatment with PapMV significantly improves effector and memory CD8(+) T cell responses generated through dendritic cell vaccination increasing protection against a Listeria monocytogenes challenge.

Medial Longitudinal Arch Angle Presents Significant Differences Between Foot Types: A Biplane Fluoroscopy Study.

The structure of the medial longitudinal arch (MLA) affects the foot's overall function and its ability to dissipate plantar pressure forces. Previous research on the MLA includes measuring the calcaneal-first metatarsal angle using a static sagittal plane radiograph, a dynamic height-to-length ratio using marker clusters with a multisegment foot model, and a contained angle using single point markers with a multisegment foot model. The objective of this study was to use biplane fluoroscopy to measure a contained MLA angle between foot types: pes planus (low arch), pes cavus (high arch), and normal arch. Fifteen participants completed the study, five from each foot type. Markerless fluoroscopic radiostereometric analysis (fRSA) was used with a three-dimensional model of the foot bones and manually matching those bones to a pair of two-dimensional radiographic images during midstance of gait. Statistically significant differences were found between barefoot arch angles of the normal and pes cavus foot types (p = 0.036), as well as between the pes cavus and pes planus foot types (p = 0.004). Dynamic walking also resulted in a statistically significant finding compared to the static standing trials (p = 0.014). These results support the classification of individuals following a physical assessment by a foot specialist for those with pes cavus and planus foot types. The differences between static and dynamic kinematic measurements were also supported using this novel method.

Skewed epithelial cell differentiation and premature aging of the thymus in the absence of vitamin D signaling.

Central tolerance of thymocytes to self-antigen depends on the medullary thymic epithelial cell (mTEC) transcription factor autoimmune regulator (Aire), which drives tissue-restricted antigen (TRA) gene expression. Vitamin D signaling regulates Aire and TRA expression in mTECs, providing a basis for links between vitamin D deficiency and autoimmunity. We find that mice lacking Cyp27b1, which cannot produce hormonally active vitamin D, display profoundly reduced thymic cellularity, with a reduced proportion of Aire+ mTECs, attenuated TRA expression, and poorly defined cortical-medullary boundaries. Markers of T cell negative selection are diminished, and organ-specific autoantibodies are present in knockout (KO) mice. Single-cell RNA sequencing revealed that loss of Cyp27b1 skews mTEC differentiation toward Ccl21+ intertypical TECs and generates a gene expression profile consistent with premature aging. KO thymi display accelerated involution and reduced expression of thymic longevity factors. Thus, loss of thymic vitamin D signaling disrupts normal mTEC differentiation and function and accelerates thymic aging.

"Can You Feel It": An Early Experience with Simulated Vibration to Recreate Glenoid Reaming.

When developing educational simulators, meaningful haptic feedback is important. To our knowledge, no shoulder arthroplasty surgical simulator exists. This study focuses on simulating vibration haptics of glenoid reaming for shoulder arthroplasty using a novel glenoid reaming simulator. Methods: We validated a novel custom simulator constructed using a vibration transducer transmitting simulated reaming vibrations to a powered nonwearing reamer tip through a 3D-printed glenoid. Validation and system fidelity were evaluated by 9 fellowship-trained shoulder surgeon experts performing a series of simulated reamings. We then completed the validation process through a questionnaire focused on experts' experience with the simulator. Results: Experts correctly identified 52% ± 8% of surface profiles and 69% ± 21% of cartilage layers. Experts identified the vibration interface between simulated cartilage and subchondral bone (77% ± 23% of the time), indicating high fidelity for the system. An interclass correlation coefficient for experts' reaming to the subchondral plate was 0.682 (confidence interval 0.262-0.908). On a general questionnaire, the perceived utility of the simulator as a teaching tool was highly ranked (4/5), and experts scored "ease of instrument manipulation" (4.19/5) and "realism of the simulator" (4.11/5) the highest. The mean global evaluation score was 6.8/10 (range 5-10). Conclusions: We examined a simulated glenoid reamer and feasibility of haptic vibrational feedback for training. Experts validated simulated vibration feedback for glenoid simulation reaming, and the results suggested that this may be a useful additional training adjuvant. Level of Evidence: Level II, prospective study.

Arthroscopy Association of Canada Position Statement on Opioid Prescription After Arthroscopic Surgery.

Background: Despite the ongoing opioid epidemic, most patients are still prescribed a significant number of opioid medications for pain management after arthroscopic surgery. There is a need for consensus among orthopaedic surgeons and solutions to aid providers in analgesic strategies that reduce the use of opioid pain medications. Purpose: This position statement was developed with a comprehensive systematic review and meta-analysis of exclusively randomized controlled trials (RCTs) to synthesize the best available evidence for managing acute postoperative pain after arthroscopic surgery. Study Design: Position statement. Methods: The Embase, MEDLINE, PubMed, Scopus, and Web of Science databases were searched from inception until August 10, 2022. Keywords included arthroscopy, opioids, analgesia, and pain, and associated variations. We included exclusively RCTs on adult patients to gather the best available evidence for managing acute postoperative pain after arthroscopic surgery. Patient characteristics, pain, and opioid data were extracted, data were analyzed, and trial bias was evaluated. Results: A total of 21 RCTs were identified related to the prescription of opioid-sparing pain medication after arthroscopic surgery. The following recommendations regarding noninvasive, postoperative pain management strategies were made: (1) multimodal oral nonopioid analgesic regimens-including at least 1 of acetaminophen-a nonsteroidal anti-inflammatory drug-can significantly reduce opioid consumption with no change in pain scores; (2) cryotherapy is likely to help with pain management, although the evidence on the optimal method of application (continuous-flow vs ice pack application) is unclear; (3) and (4) limited RCT evidence supports the efficacy of transcutaneous electrical nerve stimulation and relaxation exercises in reducing opioid consumption after arthroscopy; and (5) limited RCT evidence exists against the efficacy of transdermal lidocaine patches in reducing opioid consumption. Conclusion: A range of nonopioid strategies exist that can reduce postarthroscopic procedural opioid consumption with equivalent vocal pain outcomes. Optimal strategies include multimodal analgesia with education and restricted/reduced opioid prescription.